CHAPTER 1

POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

BHANU P. JENA Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan

1.1. INTRODUCTION secretion and membrane fusion play an important role in health and disease. Stud- Secretion and membrane fusion are funda- ies (Abu-Hamdah et al., 2004; Anderson, mental cellular processes regulating endo- 2004; Cho et al., 2002a–f, 2004; Horber¨ plasmic reticulum (ER)–Golgi transport, and Miles, 2003; Jena, 1997, 2002–2004; plasma membrane recycling, cell division, Jena et al., 1997, 2003; Jeremic et al., 2003, sexual reproduction, acid secretion, and the 2004a,b; Kelly et al., 2004; Schneider et al., release of enzymes, hormones, and neuro- 1997) in the last decade demonstrate that transmitters, to name just a few. It is there- membrane-bound secretory vesicles dock
fore no surprise that defects in secretion and and transiently fuse at the base of specialized
membrane fusion give rise to diseases such plasma membrane structures called poro- as diabetes, Alzheimer’s, Parkinson’s, acute somes or fusion pores, to expel vesicular con- gastroduodenal diseases, gastroesophageal tents. These studies further demonstrate that reflux disease, intestinal infections due to during secretion, secretory vesicles swell, inhibition of gastric acid secretion, biliary enabling the expulsion of intravesicular diseases resulting from malfunction of secre- tion from hepatocytes, polycystic ovarian contents through porosomes (Abu-Hamdah disease as a result of altered gonadotropin et al., 2004; Cho et al., 2002f; Jena et al., secretion, and Gitelman disease associated 1997; Kelly et al., 2004). With these find- with growth hormone deficiency and distur- ings (Abu-Hamdah et al., 2004; Anderson, bances in vasopressin secretion. Understand- 2004; Cho et al., 2002a–f, 2004; Horber¨ ing cellular secretion and membrane fusion and Miles, 2003; Jena, 1997, 2002–2004; not only helps to advance our understand- Jena et al., 1997, 2003; Jeremic et al., 2003, ing of these vital cellular and physiological 2004a,b; Kelly et al., 2004; Schneider et al., processes, but also helps in the develop- 1997) a new understanding of cell secre- ment of drugs to ameliorate secretory defects, tion has emerged and confirmed by a num- provides insight into our understanding of ber of laboratories (Aravanis et al., 2003; cellular entry and exit of viruses and other Fix et al., 2004; Lee et al., 2004; Taraska pathogens, and helps in the development et al., 2003; Thorn et al., 2004; Tojima et al., of smart drug delivery systems. Therefore, 2000).

Force Microscopy: Applications in Biology and Medicine, edited by Bhanu P. Jena and J.K. Heinrich Horber.¨ Copyright  2006 John Wiley & Sons, Inc. 1

2 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

Throughout history, the development of 2004; Horber¨ and Miles, 2003; Jena, 1997, new imaging tools has provided new insights 2002–2004; Jena et al., 1997, 2003; Jeremic into our perceptions of the living world and et al., 2003, 2004a,b; Kelly et al., 2004; has profoundly impacted human health. The Schneider et al., 1997) and membrane fusion invention of the light microscope almost (Cho et al., 2002d; Jeremic et al., 2004a,b; 300 years ago was the first catalyst, pro- Weber et al., 1998), as noted earlier in the pelling us into the era of modern biology chapter. and medicine. Using the light microscope, a The resolving power of the light micro- giant step into the gates of modern medicine scope is dependent on the wavelength of the was made by the discovery of the unit of light used; therefore, 250–300 nm in lateral life, the cell. The structure and morphology resolution, and much less in depth resolu- of normal and diseased cells and of disease- tion, can be achieved at best. The porosome causing microorganisms were revealed for or fusion pore in live secretory cells are cup- the first time using the light microscope. shaped structures, measuring 100–150 nm at Then in 1938, with the birth of the elec- its opening and 15–30 nm in relative depth tron microscope (EM), dawned a new era in the exocrine pancreas, and just 10 nm at in biology and medicine. Through the mid- the presynaptic membrane of the nerve ter- 1940s and 1950s, a number of subcellular minal. As a result, it had evaded visual detec- organelles were discovered and their func- tion until its discovery using the AFM (Cho tions determined using the EM. Viruses, et al., 2002a–c, 2003, 2004; Jeremic et al., the new life forms, were discovered and 2003; Schneider et al., 1997). The develop- observed for the first time and were impli- ment of the AFM (Binnig et al., 1986) has cated in diseases ranging from the com- enabled the imaging of live cells in physio- mon cold to acquired immune deficiency logical buffer at nanometer to subnanometer
syndrome (AIDS). Despite the capability resolution. In AFM, a probe tip microfab-
of the EM to image biological samples at ricated from silicon or silicon nitride and near-nanometer resolution, sample process- mounted on a cantilever spring is used to ing (fixation, dehydration, staining) results in scan the surface of the sample at a con- morphological alterations and was a major stant force. Either the probe or the sample concern. Then in the mid-1980s, scanning can be precisely moved in a raster pattern probe microscopy evolved (Binnig et al., using an xyz piezo tube to scan the sur- 1986; Horber¨ and Miles, 2003), further face of the sample (Fig. 1.1). The deflection extending our perception of the living world of the cantilever measured optically is used to the near atomic realm. One such scanning to generate an isoforce relief of the sam- probe microscope, the atomic force micro- ple (Alexander et al., 1989). Force is thus scope (AFM), has helped overcome both used to image surface profiles of objects by limitations of light and electron microscopy, the AFM, allowing imaging of live cells and enabling determination of the structure and subcellular structures submerged in physio- dynamics of single biomolecules and live logical buffer solutions. To image live cells, cells in 3D, at near-angstrom resolution. This the scanning probe of the AFM operates in unique capability of the AFM has given physiological buffers and may do so under rise to a new discipline of “nanobioscience,” two modes: contact or tapping. In the con- heralding a new era in biology and medicine. tact mode, the probe is in direct contact Using AFM in combination with conven- with the sample surface as it scans at a con- tional tools and techniques, this past decade stant vertical force. Although high-resolution has witnessed advances in our understand- AFM images can be obtained in this mode ing of cell secretion (Abu-Hamdah et al., of AFM operation, sample height informa- 2004; Anderson, 2004; Cho et al., 2002a–f, tion generated may not be accurate since

METHODS 3

Position Sensitive Photosensor Monitor Laser Source

Oscillating Cantilever

Computer

Fluid Chamber

Figure 1.1. Schematic diagram depicting key components of an atomic force microscope.

the vertical scanning force may depress the 1.2. METHODS soft cell. However, information on the vis- 1.2.1. Isolation of Pancreatic coelastic properties of the cell and the spring Acinar Cells constant of the cantilever enables measure- ment of the cell height. In tapping mode Acinar cells for secretion experiments, on the other hand, the cantilever resonates light microscopy, atomic force microscopy (AFM), and electron microscopy (EM) were and the tip makes brief contacts with the
isolated using minor modification of a
sample. In the tapping mode in fluid, lateral published procedure. For each experiment, forces are virtually negligible. It is therefore a male Sprague–Dawley rat weighing important that the topology of living cells 80–100 g was euthanized by CO2 inhalation. be obtained using both contact and tapping The pancreas was dissected and diced modes of AFM operation in fluid. The scan- into 0.5-mm3 pieces with a razor blade, ◦ ning rate of the tip over the sample also plays mildly agitated for 10 min at 37 Cin an important role on the quality of the image. a siliconized glass tube with 5 ml of Since cells are soft samples, a high scan- oxygenated buffer A (98 mM NaCl, 4.8 mM ning rate would influence its shape. Hence, KCl, 2 mM CaCl2,1.2mMMgCl2,0.1% a slow tip movement over the cell would bovine serum albumin, 0.01% soybean be ideal and results in minimal distortion trypsin inhibitor, 25 mM Hepes, pH 7.4) and better image resolution. Rapid cellular containing 1000 units of collagenase. events may be further monitored by using The suspension of acini was filtered through a 224-µm Spectra-Mesh (Spectrum section analysis. To examine isolated cells by Laboratory Products, Rancho Dominguez, the AFM, freshly cleaved mica coated with CA) polyethylene filter to remove large Cel-Tak have also been used with great suc- clumps of acini and undissociated tissue. The cess (Cho et al., 2002a–c; Jena et al., 2003; acini were washed six times, 50 ml per wash, Jeremic et al., 2003; Schneider et al., 1997). with ice-cold buffer A. Isolated rat pancreatic Also, to obtain optimal resolution, the con- acini and acinar cells were plated on tents of the bathing medium as well as the Cell-Tak-coated (Collaborative Biomedical cell surface to be scanned should be devoid Products, Bedford, MA) glass coverslips. of any debris. Two to three hours after plating, cells were

4 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

imaged with the AFM before and during supplemented with protease inhibitor cock- stimulation of secretion. Isolated acinar cells tail (Sigma, St. Louis, MO) and homoge- and hemi-acinar preparations were used in nized using Teflon-glass homogenizer (8–10 the study because fusion of secretory vesicles strokes). The total homogenate was cen- at the PM in these cells occurs at the apical trifuged for 3 min at 2500 × g. The super- region facing the acinar lumen. natant fraction was further centrifuged for 15 min at 14,500 × g, and the resultant pellet 1.2.2. Pancreatic Plasma Membrane was resuspended in buffered sucrose solu- Preparation tion, which was loaded onto 3–10–23% Percoll gradients. After centrifugation at Rat pancreatic PM fractions were iso- 28,000 × g for 6 min, the enriched synapto- lated using a modification of a published somal fraction was collected at the 10–23% method. Male Sprague–Dawley rats weigh- Percoll gradient interface. To isolate synaptic ing 70–100 g were euthanized by CO2 vesicles and synaptosomal membrane (32), inhalation. Pancreas were removed and isolated synaptosomes were diluted with 9 placed in ice-cold phosphate-buffered saline vol of ice-cold H2O (hypotonic lysis of (PBS), pH 7.5. Adipose tissue was removed synaptosomes to release synaptic vesicles) 3 and the pancreas were diced into 0.5-mm and immediately homogenized with three pieces using a razor blade in a few drops strokes in Dounce homogenizer, followed by of homogenization buffer A (1.25 M sucrose, a 30-min incubation on ice. The homogenate 0.01% trypsin inhibitor, and 25 mM Hepes, was centrifuged for 20 min at 25,500 × g, pH 6.5). The diced tissue was homogenized and the resultant pellet (enriched synaptoso- in 15% (w/v) ice-cold homogenization buffer mal membrane preparation) and supernatant A using four strokes at maximum speed (enriched synaptic vesicles preparation) were of a motor-driven pestle (Wheaton over- used in our studies.
head stirrer). One-and-a-half milliliters of the
homogenate was layered over a 125-µl cush- ion of 2 M sucrose and 500 µlof0.3M 1.2.4. Preparation of Lipid Membrane sucrose was layered onto the homogenate in on Mica and Porosome Reconstitution Beckman centrifuge tubes. After centrifuga- To prepare lipid membrane on mica for tion at 145,000 × g for 90 min in a Sorvall AFM studies, freshly cleaved mica disks AH-650 rotor, the material banding between were placed in a fluid chamber. Two hundred the 1.2 and 0.3 M sucrose interface was microliters of the bilayer bath solution, collected and the protein concentration was containing 140 mM NaCl, 10 mM HEPES, determined. For each experiment, fresh PM and 1 mM CaCl , was placed at the center of was prepared and used the same day in all 2 the cleaved mica disk. Ten microliters of the AFM experiments. brain lipid vesicles was added to the above bath solution. The mixture was then allowed 1.2.3. Isolation of Synaptosomes, to incubate for 60 min at room temperature, Synaptosomal Membrane and before washing (×10), using 100-µlbath Synaptic Vesicles solution/wash. The lipid membrane on mica Synaptosomes, synaptosomal membrane and was imaged by the AFM before and after the synaptic vesicles were prepared from rat addition of immunoisolated porosomes. brains (Jeong et al., 1998; Thoidis et al., 1998). Whole rat brain from Sprague– 1.2.5. Atomic Force Microscopy Dawley rats (100–150 g) was isolated and placed in ice-cold buffered sucrose solu- “Pits” and fusion pores at the PM in live tion (5 mM Hepes pH 7.4, 0.32 M sucrose) pancreatic acinar secreting cells in PBS pH

METHODS 5

7.5 were imaged with the AFM (Bioscope 1.2.8. Isolation of Zymogen Granules III, Digital Instruments) using both contact ZGs were isolated by using a modifica- and tapping modes. All images presented tion of the method of our published proce- in this manuscript were obtained in the dure. Male Sprague–Dawley rats weighing “tapping” mode in fluid, using silicon nitride 80–100 g were euthanized by CO inhala- tips with a spring constant of 0.06 N· 2 − tion for each ZG preparation. The pancreas m 1 andanimagingforceof<200 pN. was dissected and diced into 0.5-mm3 pieces. Images were obtained at line frequencies of The diced pancreas was suspended in 15% 1 Hz, with 512 lines per image and constant (w/v) ice-cold homogenization buffer (0.3 M image gains. Topographical dimensions of sucrose, 25 mM Hepes, pH 6.5, 1 mM ben- “pits” and fusion pores at the cell PM zamidine, 0.01% soybean trypsin inhibitor) were analyzed using the software nanoscope and homogenized with a Teflon glass homog- IIIa4.43r8 supplied by Digital Instruments. enizer. The resultant homogenate was cen- ◦ trifuged for 5 min at 300 × g at 4 C to obtain a supernatant fraction. One volume of the 1.2.6. ImmunoAFM on Live Cells supernatant fraction was mixed with 2 vol Immunogold localization in live pancreatic of a Percoll–Sucrose–Hepes buffer (0.3 M acinar cells was assessed after a 5-min sucrose, 25 mM Hepes, pH 6.5, 86% Per- stimulation of secretion with 10 µMofthe coll, 0.01% soybean trypsin inhibitor) and centrifuged for 30 min at 16,400 × g at secretagogue, mastoparan. After stimulation ◦ of secretion, the live pancreatic acinar cells 4 C. Pure ZGs were obtained as a loose in buffer were exposed to at 1:200 dilution white pellet at the bottom of the centrifuge of α-amylase-specific antibody (Biomeda tube. Corp., Foster City, CA) and 30 nm of gold
conjugated secondary antibody for 1 min and 1.2.9. Transmission Electron
were washed in PBS before AFM imaging Microscopy in PBS at room temperature. “Pits” and fusion pores within, at the apical end of live Isolated rat pancreatic acini and ZGs were pancreatic acinar cells in PBS pH 7.5, were fixed in 2.5% buffered paraformaldehyde imaged by the AFM (Bioscope III, Digital (PFA) for 30 min, and the pellets were Instruments) using both contact and tapping embedded in Unicryl resin and were sec- mode. All images presented were obtained tioned at 40–70 nm. Thin sections were in the “tapping” mode in fluid, using silicon transferred to coated specimen TEM grids, nitride tips as described previously. dried in the presence of uranyl acetate and methyl cellulose, and examined in a trans- mission electron microscope. 1.2.7. ImmunoAFM on Fixed Cells 1.2.10. Immunoprecipitation and After stimulation of secretion with 10 µM Western Blot Analysis mastoparan, the live pancreatic acinar cells were fixed for 30 min using ice-cold 2.5% Immunoblot analysis was performed on pan- paraformaldehyde in PBS. Cells were then creatic PM and total homogenate fractions. washed in PBS, followed by labeling with Protein in the fractions was estimated by the 1:200 dilution of α-amylase-specific anti- Bradford method (Bradford, 1976). Pancre- body (Biomeda Corp.) and 10 nm of gold atic fractions were boiled in Laemmli reduc- conjugated secondary antibody for 15 min, ing sample preparation buffer (Laemmli, fixed, washed in PBS, and imaged in PBS at 1970) for 5 min, cooled, and used for SDS- room temperature using the AFM. PAGE. PM proteins were resolved in a 12.5%

6 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

SDS-PAGE and electrotransferred to 0.2-µm 1.3. POROSOME: A NEW nitrocellulose sheets for immunoblot analy- CELLULAR STRUCTURE sis with a SNAP-23 specific antibody. The nitrocellulose was incubated for 1 h at room Earlier electrophysiological studies on mast temperature in blocking buffer (5% non- cells suggested the existence of fusion pores fat milk in PBS containing 0.1% Triton X- at the cell plasma membrane (PM), which 100 and 0.02% NaN3), and immunoblot- became continuous with the secretory vesicle ted for 2 h at room temperature with the membrane following stimulation of secretion SNAP-23 antibody (ABR, Golden, CO). The (Monck et al., 1995). AFM has confirmed primary antibodies were used at a dilu- the existence of the fusion pore or porosome as permanent structures at the cell plasma tion of 1:10,000 in blocking buffer. The membrane and has revealed its morphol- immunoblotted nitrocellulose sheets were ogy and dynamics in the exocrine pancreas washed in PBS containing 0.1% Triton X- (Cho et al., 2002a; Jena et al., 2003; Jeremic 100 and 0.02% NaN3 and were incubated for et al., 2003; Schneider et al., 1997), neuroen- 1 h at room temperature in HRP-conjugated docrine cells (Cho et al., 2002b,c), and neu- secondary antibody at a dilution of 1:2,000 rons (Cho et al., 2004), at near-nanometer in blocking buffer. The immunoblots were resolution and in real time. then washed in the PBS buffer, processed for Isolated live pancreatic acinar cells in enhanced chemiluminescence, and exposed physiological buffer, when imaged with the to X-OMAT-AR film. To isolate the fusion AFM (Cho et al., 2002a; Jena et al., 2003; complex for immunoblot analysis, SNAP- Jeremic et al., 2003; Schneider et al., 1997), 23 specific antibody conjugated to pro- reveal at the apical PM a group of circu- µ tein A-sepharose was used. One gram of lar “pits” measuring 0.4–1.2 mindiam-
eter which contain smaller ‘depressions’
total pancreatic homogenate solubilized in (Fig. 1.2). Each depression averages between Triton/Lubrol solubilization buffer (0.5% 100 and 150 nm in diameter, and typically Lubrol; 1 mM benzamidine; 5 mM ATP; 3–4 depressions are located within a pit. 5 mM EDTA; 0.5% Triton X-100, in PBS) The basolateral membrane of acinar cells is supplemented with protease inhibitor mix devoid of either pits or depressions. High- (Sigma, St. Louis, MO) was used. SNAP- resolution AFM images of depressions in 23 antibody conjugated to the protein A- live cells further reveal a cone-shaped mor- sepharose was incubated with the solubilized phology. The depth of each depression cone homogenate for 1 h at room temperature measures 15–30 nm. Similarly, growth hor- followed by washing with wash buffer mone (GH)-secreting cells of the pituitary (500 mM NaCl, 10 mM TRIS, 2 mM EDTA, gland and chromaffin cells, β cells of the pH = 7.5). The immunoprecipitated sample exocrine pancreas, mast cells, and neurons attached to the immuno-sepharose beads was possess depressions at their PM, suggesting incubated in Laemmli sample preparation their universal presence in secretory cells. Exposure of pancreatic acinar cells to a sec- buffer—prior to 12.5% SDS-PAGE, electro- retagogue (mastoparan) results in a time- transfer to nitrocellulose, and immunoblot dependent increase (20–35%) in depression analysis—using specific antibodies to actin diameter, followed by a return to resting (Sigma), fodrin (Santa Cruz Biotechnology size on completion of secretion (Cho et al., Inc., Santa Cruz, CA), vimentin (Sigma, 2002a; Jena et al., 2003; Jeremic et al., 2003; St. Louis, MO), syntaxin 2 (Alomone Labs, Schneider et al., 1997) (Fig. 1.3). No demon- + Jerusalem, Israel), Ca2 -β 3 (Alomone Labs), strable change in pit size is detected follow- and Ca2+-α 1c (Alomone Labs). ing stimulation of secretion (Schneider et al.,

POROSOME: A NEW CELLULAR STRUCTURE 7

Figure 1.2. (A) On the far left is an AFM micrograph depicting “pits” and “depressions” within, at the plasma membrane in live pancreatic acinar cells. On the right is a schematic drawing depicting depressions, at the cell plasma membrane, where membrane-bound secretory vesicles dock and fuse to release vesicular contents (Schneider et al., 1997). (B) Electron micrograph depicting a porosome close to a microvilli (MV) at the apical plasma membrane (PM) of a pancreatic acinar cell. Note association of the porosome membrane and the zymogen granule membrane (ZGM) of a docked zymogen granule (ZG), the membrane-bound secretory vesicle of exocrine pancreas. Also a cross section of the ring at the mouth of the porosome is seen.

8 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

Figure 1.3. Dynamics of depressions following stimulation of secretion. The top panel shows a number of depressions within a pit in a live pancreatic acinar cell. The scan line across three depressions in the top panel is represented graphically in the middle panel and defines the diameter and relative depth of the depressions; the middle depressions are represented by arrowheads. The bottom panel represents percent of total cellular amylase release in the presence and absence of the secretagogue Mas 7. Notice an increase in the diameter and depth of depressions, correlating with an increase in total cellular amylase release at 5 min after stimulation of secretion. At 30 min after stimulation of secretion, there is a decrease in diameter and depth of depressions, with no further

increase in amylase release over the 5-min time point. No significant increase in amylase secretion or depressions diameter were observed in resting acini or those exposed to the nonstimulatory mastoparan analog Mas 17 (Jena, 2002; Schneider et al., 1997).

1997). Enlargement of depression diameter also reveal the presence of pits and depres- and an increase in its relative depth after sions at the cell PM (Fig. 1.4). The presence exposure to secretagogues correlated with of porosomes in neurons, in β cells of the increased secretion. Conversely, exposure of endocrine pancreas, and in mast cells have pancreatic acinar cells to cytochalasin B, a also been demonstrated (Figs. 1.4–1.8) (Cho fungal toxin that inhibits actin polymeriza- et al., 2004; Jena, 2004). Depressions in rest- ± ± tion, results in a 15–20% decrease in depres- ing GH cells measure 154 4.5nm(mean sion size and a consequent 50–60% loss SE) in diameter. Exposure of GH cells to a secretagogue results in a 40% increase in secretion (Schneider et al., 1997). Results in depression diameter (215 ± 4.6nm;p< from these studies suggested depressions to 0.01) but no appreciable change in pit be the fusion pores in pancreatic acinar cells. size. The enlargement of depression diam- Furthermore, these studies demonstrate the eter during secretion and the known effect involvement of actin in regulation of both the that actin depolymerizing agents decrease structure and function of depressions. Anal- depression size and inhibit secretion (Schnei- ogous to pancreatic acinar cells, examination der et al., 1997) suggested depressions to of resting GH-secreting cells of the pitu- be the fusion pores. However, a more itary (Cho et al., 2002b) and chromaffin cells direct demonstration that porosomes are of the adrenal medulla (Cho et al., 2002c) functional supramolecular complexes came

POROSOME: A NEW CELLULAR STRUCTURE 9

A B

C D

Figure 1.4. AFM micrograph of depressions or porosomes or fusion pores in the live secretory cell of the exocrine pancreas (A, B), the growth hormone (GH)-secreting cell of the pituitary (C), and in the chromaffin cell (D). Note
the “pit” (white arrowheads at the margin) with four depressions (arrowhead at 12 o’ clock). A high-resolution AFM
micrograph of one porosome is shown in part B. Bars = 40 nm for parts A and B. Similarly, AFM micrographs of porosomes in β cell of the endocrine pancreas and mast cell have also been demonstrated.

Figure 1.5. Porosomes or fusion pores in β cells of the endocrine pancreas. (A) AFM micrograph of a pit with three porosomes (arrowheads) at the PM in a live β cell. (B) Section analysis through a porosome at rest (top trace), during secretion (bottom trace), and following completion of secretion (middle trace). (C) Exposure of β cells to the actin depolymerizing agent cytochalasin B results in decreased “porosome” size (not shown), accompanied by a loss in insulin secretion, as detected by immunoblot analysis of the cell incubation medium.

10 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

Figure 1.6. Electron micrograph of porosomes in neurons. (A) Electron micrograph of a synaptosome demonstrating the presence of 40- to 50-nm synaptic vesicles. (B–D) Electron micrograph of neuronal porosomes, which are 10- to 15-nm cup-shaped structures at the presynaptic membrane (arrowhead), where synaptic vesicles transiently dock and fuse to release vesicular contents. (E) Atomic force micrograph of a fusion pore or porosome at the nerve terminal in a live synaptosome. (F) Atomic force micrograph of isolated neuronal porosome, reconstituted into lipid membrane.

from immuno-AFM studies demonstrating an increase in capacitance and conductance the specific localization of secretory prod- and in a time-dependent release of the ZG ucts at the porosomes, following stimula- enzyme amylase from cis to the trans com- tion of secretion (Figs. 1.9–1.12) (Jeremic partment of the bilayer chamber. Amylase is et al., 2003). ZGs fused with the porosome- detected using immunoblot analysis of the reconstituted bilayer, as demonstrated by buffer in the cis and trans chambers, using

POROSOME: A NEW CELLULAR STRUCTURE 11

Resting Stimulated Amylase Ab-gold Fixed Amylase Ab-gold ABCD

nm nm nm

µm 2.0

0 0 0 1.5 1.0 0.5 75 75 75 75 75 75

− 0 0.2 0.4 0.6 0.8 0− 0.2 0.4 0.6 0.8 0− 0.2 0.4 0.6 0.8 µm µm µm

Figure 1.7. Depressions are fusion pores or porosomes. Porosomes dilate to allow expulsion of intra vesicular contents. (A, B) AFM micrographs and section analysis of a pit and two out of the four fusion pores or porosomes, demonstrating enlargement following stimulation of secretion. (C) Exposure of live cells to gold conjugated-amylase antibody (Ab) results in specific localization of immuno-gold to the porosome opening. (arrowhead) Amylase is one of the proteins within secretory vesicles of the exocrine pancreas. (D) AFM micrograph of a fixed pancreatic acinar cell, demonstrating a pit and porosomes within labeled with amylaze-immunogold. Arrowheads point to immunogold clusters (Cho et al., 2002a) at the pit and porosomes within.

a previously characterized amylase specific determined by sypro protein staining of antibody (Cho et al., 2002a). As observed the resolved complex (Fig. 1.13A). Fur- in immunoblot assays of isolated porosomes, thermore, electrotransfer of the resolved chloride channel activities are also detected porosomal complex onto nitrocellulose mem-
within the reconstituted porosome complex. brane, followed by immunoblot analy-
Furthermore, the chloride channel inhibitor sis using various antibodies, revealed 9 DIDS was found to inhibit current activ- proteins. In agreement with earlier find- ity in the porosome-reconstituted bilayer. ings, SNAP-25, the P/Q-type calcium In summary, these studies demonstrate that channel, actin, syntaxin-1, synaptotagmin- the porosome in the exocrine pancreas is 1, vimentin, the N-ethylmaleimide-sensitive a 100- to 150-nm-diameter supramolecular factor (NSF), the chloride channel CLC-3, cup-shaped lipoprotein basket at the cell PM, and the alpha subunit of the heterotrimeric where membrane-bound secretory vesicles GTP-binding Go were identified to con- dock and fuse to release vesicular contents. stitute part of the complex. To test Similar studies have now been performed whether the complete porosome complex in neurons, demonstrating both the struc- was immunoisolated, the immunoisolate tural (Fig. 1.6E,F) and functional reconstitu- was reconstituted into lipid membrane tion of the isolated neuronal porosome com- prepared using brain dioleoylphosphatidyl- plex. The biochemical composition of the choline (DOPC) and dioleylphosphatidylser- neuronal porosome has also been determined ine (DOPS) in a ratio of 7:3. At low (Cho et al., 2004). resolution, the AFM reveals the immunoiso- Similar to the isolation of porosomes lates to arrange in L- or V-shaped struc- from the exocrine pancreas, neuronal poro- tures (Fig. 1.13B, red arrowheads), which somes were immunoisolated from detergent- at higher resolution demonstrates the pres- solubilized synaptosome preparations, using ence of porosomes in patches (Fig. 1.13C,D, a SNAP-25 specific antibody. Electrophoretic green arrowhead), similar to what is observed resolution of the immunoisolates reveal the at the presynaptic membrane in intact synap- presence of 12 distinct protein bands, as tosomes. Further imaging the reconstituted

12 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

Figure 1.8. Morphology of the cytosolic side of the porosome revealed in AFM studies on isolated pancreatic plasma membrane (PM) preparations. (A) AFM micrograph of isolated PM preparation reveals the cytosolic end of a pit with inverted cup-shaped structures, the porosome. Note the 600 nm in diameter ZG at the left-hand corner of the pit. (B) Higher magnification of the same pit showing clearly the 4 to 5 porosomes within. (C) The cytosolic end of a single porosome is depicted in this AFM micrograph. (D) Immunoblot analysis of 10 µg and 20 µgof pancreatic PM preparations, using SNAP-23 antibody, demonstrates a single 23-kDa immunoreactive band. (E, F) The cytosolic side of the PM demonstrating the presence of a pit with a number of porosomes within, shown prior to (E) and following addition of the SNAP-23 antibody (F) Note the increase in height of the porosome cone base revealed by section analysis (bottom panel), demonstrating localization of SNAP-23 antibody at the base of the porosome (Jeremic et al., 2003).

immunoisolate at greater resolutions using cytosolic side of the presynaptic membrane, the AFM shows the presence of 8- to 10- isolated synaptosomal membrane prepara- nm porosomes (Fig. 1.13E). As observed tions were imaged by the AFM. These in electron and AFM micrographs of the studies reveal the architecture of the cytoso- presynaptic membrane in synaptosomes, the lic part of porosomes. In synaptosomes, immunoisolated and lipid-reconstituted poro- 40- to 50-nm synaptic vesicles are arranged some reveals the presence of an approxi- in ribbons, as seen in the electron micro- mately 2 nm in diameter central plug. These graphs (longitudinal section, Fig. 1.14A, or studies confirm the complete isolation and in cross section Fig. 1.14B). Similarly, when structural reconstitution of the neuronal poro- the cytosolic domain of isolated synaptoso- some in artificial lipid bilayers. To under- mal membrane preparations were analyzed stand the structure of the porosome at the by the AFM, 40- to 50-nm synaptic vesicles

POROSOME: A NEW CELLULAR STRUCTURE 13

Figure 1.9. SNAP-23 associated proteins in pancreatic acinar cells. Total pancreatic homogenate was immunopre- cipitated using the SNAP-23 specific antibody. The precipitated material was resolved using 12.5% SDS-PAGE, electrotransferred to nitrocellulose membrane and then probed using antibodies to a number of proteins. Association of SNAP-23 with syntaxin2, with cytoskeletal proteins actin, α-fodrin, and vimentin, and calcium channels β3and α1c, together with the SNARE regulatory protein NSF, is demonstrated (arrowheads). Lanes showing more than one
arrowhead suggest presence of isomers or possible proteolytic degradation of the specific protein (Jena et al., 2003).

were also found to be arranged in ribbons involved in fusion of opposing bilayers (Fig. 1.14C,D), thus confirming EM observa- (Jeremic et al., 2004a,b). NSF is an ATPase tions. On close examination (Fig. 1.14D–F) that is suggested to disassemble the t-/v- of docked synaptic vesicles (blue arrow- SNARE complex in the presence of ATP heads), synaptic vesicles are found attached (Jeong et al., 1998). To test this hypothe- to the base of the 8- to 10-nm porosomes (red sis and to further confirm and determine the arrowheads). It is possible to see both the morphology of neuronal porosomes, synap- porosome and the attached synaptic vesicle, tosomal membrane preparations with docked since while imaging with the AFM, synap- synaptic vesicles were imaged using the tic vesicles can be gently pushed away from AFM in the presence and absence of ATP their docked sites to reveal the porosome (Fig. 1.15A). As hypothesized, addition of lying beneath them (Fig. 1.14C–F). Addi- 50 µM ATP resulted in t-/v-SNARE dis- tionally, bare porosomes with no synaptic assembly and the release of docked vesi- vesicles attached are also found at the cytoso- cles (blue arrowhead) at the porosome patch lic side of synaptosomal membrane prepara- (Fig. 1.15B,C, red arrowhead). At higher tions (Fig. 1.14D). resolution, the base of the porosome is Calcium, target membrane proteins clearly revealed (Fig. 1.15D). At increased SNAP-25 and syntaxin (t-SNARE), and imaging forces (300–500 pN instead of secretory vesicle-associated membrane pro- <200 pN), porosome patches (Fig. 1.15E, tein (v-SNARE) are the minimal machinery red arrowhead) and individual porosomes

14 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

Figure 1.10. Negatively stained electron micrograph and atomic force micrograph of the immunoisolated porosome complex. (A) Negatively stained electron micrograph of an immunoisolated porosome complex from solubilized pancreatic plasma membrane preparations, using a SNAP-23 specific antibody. Note the three rings and the 10 spokes
that originate from the inner smallest ring. This structure represents the protein backbone of the porosome complex,
since the three rings and the vertical spikes are observed in electron micrographs of cells and porosome co-isolated with ZGs. Bar = 30 nm. (B) Electron micrograph of the fusion pore complex, cut out from (A). (C) Outline of the structure presented for clarity. (D–F) Atomic force micrograph of the isolated pore complex in near physiological buffer. Bar = 30 nm. Note the structural similarity of the complex, imaged both by EM (G)andAFM(H). The EM and AFM micrographs are superimposable (I).

Figure 1.11. Electron micrographs of reconstituted porosome or fusion pore complex in liposomes, showing a cup-shaped basket-like morphology. (A) A 500-nm vesicle with an incorporated porosome is shown. Note the spokes in the complex. (B–D) The reconstituted complex at greater magnification is shown. Bar represents 100 nm.

POROSOME: A NEW CELLULAR STRUCTURE 15

A

+−

Bilayer EPC 9 Amplifier

B

Capacitance 400 Current 10 ZG Addition

pF 200 5 pA

0 0 0369 min

Cis Trans C 0 30 min

− Cl Channel − Cl Blocker

Pore Complex Pore Complex + 20 µM DIDS

1.0

nA 0.5

0 05 10 msec

Figure 1.12. Lipid bilayer-reconstituted porosome complex is functional. (A) Schematic drawing of the EPC9 bilayer setup for electrophysiological measurements. (B) Zymogen granules (ZGs) added to the cis compartment of the bilayer chamber fuse with the reconstituted porosomes, as demonstrated by an increase in capacitance and current activities, and a concomitant time-dependent release of amylase (a major ZG content) to the trans compartment of the bilayer chamber. The movement of amylase from the cis to the trans side of the chamber was determined by immunoblot analysis of the contents in the cis and the trans chamber over time. (C) As demonstrated by immunoblot analysis of the immunoisolated complex, electrical measurements in the presence and absence of chloride ion channel blocker DIDS demonstrate the presence of chloride channels in association with the complex, and its role in porosome function.

16 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

A

BC

D E

Figure 1.13. Composition of the neuronal porosome and its reconstitution in lipid membrane. (A) Proteins immunoisolated from detergent-solubilized synaptosomal membrane preparation, using a SNAP-25 specific antibody. Immunoisolates when resolved by SDS-PAGE, along with the resolved proteins in the gel stained using Sypro dye, reveals 12 specific bands (•), suggesting the presence of at least 12 proteins in the complex, not including the heavy chain (*) of the SNAP-25 antibody. When the resolved proteins were electrotransferred to nitrocellulose membrane and probed with various antibodies, calcium channel P/Q, actin, Gαo, syntaxin-1 (Syn1), synaptotagmin-1 (Syt1), NSF, vimentin, and the chloride channel CLC-3 were identified (Bradford, 1976; Laemmli, 1970). (B–E) Atomic force micrographs at different resolution of the reconstituted immunoisolate in lipid membrane. (B) At low magnification, the immunoisolated complex arrange in L- or V-shaped structures (white arrowheads). (C, D) Within the V-shaped structures, patches (arrowheads) of porosomes are found. (E) Each reconstituted porosome is almost identical to the porosome observed in intact synaptosomes. The central plug is clearly seen. This AFM micrograph demonstrates the presence of two porosomes (arrowhead), although only half of the second porosome is in view.

MOLECULAR UNDERSTANDING OF CELL SECRETION 17

(Fig. 1.15F,G) were further defined in greater synaptic vesicle proteins SV2 and VAMP-2 structural detail. At 5–8 A˚ resolution, eight (Fig. 1.16E). Our study demonstrates that peripheral knobs at the porosomal opening both SV2 and VAMP-2 proteins are enriched (Fig. 1.15F–H, yellow arrowheads) and a in supernatant fractions following exposure central plug (green arrowhead) at the base of isolated synaptosomal membrane to ATP were revealed. These studies further provide (Fig. 1.16E). Thus, AFM, electrophysiologi- a direct demonstration of synaptic vesicles cal measurements, and immunoanalysis con- docking at these sites and confirming them firmed the dissociation of porosome-docked to be porosomes, where synaptic vesicles synaptic vesicles following ATP exposure. dock and transiently fuse to release neuro- As previously suggested (Jena, 1997, 2002), transmitters (Aravanis et al., 2003). Hence such a mechanism may allow for the multiple synaptic vesicles are able to fuse transiently transient docking-fusion and release cycles and successively at porosomes in the presy- that synaptic vesicles may undergo during naptic membrane without loss of identity, as neurotransmission, without loss of vesicle reported in earlier studies (Aravanis et al., identity (Aravanis et al., 2003). The neuronal 2003). porosome, although an order of magnitude To be able to assess the functionality smaller than those in the exocrine pancreas of the reconstituted porosome preparations or in neuroendocrine cells, possesses many (Figs. 1.13E and 1.16A), an electrophysio- similarities both in structure and composi- logical bilayer setup was used (Fig. 1.16B). tion. Thus, nature has designed the porosome Membrane capacitance was continually as a general secretory machinery, but has monitored throughout these experiments. fine-tuned it to suit various secretory pro- Following reconstitution of the bilayer mem- cesses in different cells. Hence, porosome brane with porosomes, isolated synaptic vesi- size may be a form of such fine-tuning. It is well known that smaller vesicles fuse more
cles were added to the cis compartment
of the bilayer chamber. A large number efficiently than larger ones, and hence the of synaptic vesicles fused at the bilayer, curvature of both the secretory vesicle and as demonstrated by the significant step- the porosome base would dictate the effi- wise increases in the membrane capacitance cacy and potency of vesicle fusion at the (Fig. 1.16C,D). As expected from results in cell plasma membrane. For example, because Fig. 15B, addition of 50 µM ATP allowed neurons are fast secretory cells, they pos- t-/v-SNARE disassembly and the release of sess small (40–50 nm) secretory vesicles and docked vesicles, resulting in the return of porosome bases (2–4 nm) for rapid and effi- the bilayers membrane capacitance to resting cient fusion. In contrast, a slow secretory cell levels (Fig. 1.16C,D). Addition of recombi- like the exocrine pancreas possesses larger nant NSF had no further effect on mem- secretory vesicles (1000 nm in diameter) that brane capacitance. Thus, the associated NSF fuse at porosomes having bases that measure at the t-/v-SNARE complex is adequate 20–30nmindiameter. for complete disassembly of the SNARE complex for release of synaptic vesicles following transient fusion and the comple- 1.4. MOLECULAR UNDERSTANDING tion of a round of neurotransmitter release. OF CELL SECRETION To further biochemically assess the release of docked synaptic vesicles following ATP Fusion pores or porosomes as permanent treatment, synaptosomal membrane prepa- structures at the cell plasma membrane rations were exposed to 50 µMATP,and are present in all secretory cells exam- the supernatant fraction was assessed for ined, ranging from exocrine, endocrine, synaptic vesicles by monitoring levels of the and neuroendocrine cells to neurons, where

18 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

A B

CD

EF

Figure 1.14

MOLECULAR UNDERSTANDING OF CELL SECRETION 19

membrane-bound secretory vesicles dock transient fusion is the presence of neurotrans- and transiently fuse to expel vesicular con- mitter transporters at the synaptic vesicle tents. Porosomes in pancreatic acinar or GH- membrane. These vesicle-associated trans- secreting cells are cone-shaped structures porters would be of little use if vesicles at the plasma membrane, with a 100- to were to fuse completely at the plasma mem- 150-nm-diameter opening. Membrane-bound brane to be compensatorily endocytosed at secretory vesicles ranging in size from 0.2 a later time. In further support, a recent to 1.2 µm in diameter dock and fuse at study reports that single synaptic vesicles porosomes to release vesicular contents. Fol- fuse transiently and successively without loss lowing fusion of secretory vesicles at poro- of vesicle identity (Aravanis et al., 2003). somes, only a 20–35% increase in porosome Although the fusion of secretory vesicles at diameter is demonstrated. It is therefore rea- the cell plasma membrane occurs transiently, sonable to conclude that secretory vesicles complete incorporation of membrane at the “transiently” dock and fuse at the site. In cell plasma membrane would occur when contrast to accepted belief, if secretory vesi- cells need to incorporate signaling molecules cles were to completely incorporate at poro- like receptors, second messengers, or ion somes, the PM structure would distend much channels. Similarly, total fusion would occur wider than what is observed. Furthermore, if intracellularly, where during protein trans- secretory vesicles were to completely fuse port and maturation, vesicles derived from at the plasma membrane, there would be a the cis Golgi would completely fuse with loss in vesicle number following secretion. the trans Golgi apparatus. The discovery of Examination of secretory vesicles within the porosome, along with an understand- cells before and after secretion demonstrates ing of the molecular mechanism of mem- that the total number of secretory vesicles brane fusion and the swelling of secretory
remains unchanged following secretion (Cho vesicles required for expulsion of vesicu-
et al., 2002e; Lee et al., 2004). However, the lar contents, provides an understanding of number of empty and partially empty vesi- secretion and membrane fusion in cells at cles increases significantly, supporting the the molecular level. These findings have occurrence of transient fusion. Earlier studies prompted many laboratories to work in the on mast cells also demonstrated an increase area and further confirm these findings. Thus, in the number of spent and partially spent the porosome is a supramolecular structure vesicles following stimulation of secretion, universally present in secretory cells, from without any demonstrable increase in cell the exocrine pancreas to the neurons, and size. Similarly, secretory granules are recap- in the endocrine to neuroendocrine cells, tured largely intact after stimulated exocy- where membrane-bound secretory vesicles tosis in cultured endocrine cells (Taraska transiently dock and fuse to expel intravesic- et al., 2003). Other support in evidence of ular contents. Hence, the secretory process in

Figure 1.14. Arrangement of synaptic vesicles and porosomes at the presynaptic membrane. (A) Electron micrograph of rat brain synaptosome demonstrating the ribbon-like arrangement (inset) of 40- to 50-nm synaptic vesicles. (B) Cross section of such a ribbon (inset) reveals the interaction between the synaptic vesicles. (C, D) Examination of the presynaptic membrane from the cytosolic side (inside out) using AFM, confirmed such a ribbon arrangement of docked synaptic vesicles (white arrowheads) at porosomes (grey arrowheads). Bare porosomes (lacking docked synaptic vesicles) are also seen. The AFM micrograph in part c is a 2-D image, and the one in part d is a 3-D image. (E, F) AFM micrograph of a docked synaptic vesicle at a porosome. During imaging using the AFM, the interaction of the cantilever tip with the sample sometimes resulted in pushing away the docked synaptic vesicle, enough to expose the porosome lying beneath. AFM section analysis further reveals the size of synaptic vesicles (white section line and arrowheads) and porosomes (grey section line and arrowheads).

20 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

AB

CD

EF

Figure 1.15. AFM micrographs revealing the dynamics of docked synaptic vesicles at porosomes and the porosome architecture at 4–5 A˚ resolution. (A) AFM micrograph of five docked synaptic vesicle at porosomes. (B) Addition of 50 µM ATP dislodges two synaptic vesicles at the lower left, and exposing the porosome patches. This also demonstrates that a single synaptic vesicle may dock at more than one porosome complex. (C–G) AFM micrographs obtained at higher imaging forces (300–500 pN rather than <200 pN) reveal porosomes architecture at greater detail. (C) AFM micrograph of one of the porosome patches where a synaptic vesicle was docked prior to ATP exposure. (D) Base of a single porosome. (E) High-force AFM micrograph of the cytosolic face of the presynaptic membrane, demonstrating the ribbon arrangement of porosome patches (grey arrowhead) and docked synaptic vesicles (white arrowheads). Note how the spherical synaptic vesicles are compressed and flattened at higher imaging forces. (F, G) At such higher imaging forces, porosomes reveal the presence of eight globular structures (white arrowhead) surrounding a central plug (grey arrowhead), as demonstrated in the (H) schematic diagram.

MOLECULAR UNDERSTANDING OF CELL SECRETION 21

G H

Figure 1.15. (continued)

A B

C

D E

Figure 1.16. Functional reconstitution of immunoisolated nuronal porosomes. (A) Schematic representation of a porosome at the presynaptic membrane, with a docked synaptic vesicle (SV) at its base. (B) Schematic drawing of an EPC9 electrophysiological bilayers apparatus, to continually monitor changes in the capacitance of porosome-reconstituted membrane, when synaptic vesicles are introduced into the cis bilayers chamber followed by ATP and purified recombinant NSF protein. (C) Schematic representation of SV docking at the base of a porosome, fusing to release its contents, and disengaging in the presence of ATM. (D) Capacitance measurements of porosome-reconstituted bilayers support the experiment in Fig. 1.15B and the schematic diagram in part c. Exposure of the reconstituted bilayers to SVs results in a dramatic increase in membrane capacitance, which drops to baseline following exposure to 50 µM ATP. Recombinant NSF has no further effect (n = 6). (E) Similarly, in agreement, exposure of isolated synaptosomal membrane preparations to 50 µM ATP results in the release of SVs from the membrane into the incubation medium, as demonstrated by immunoblot analysis of the incubating medium using the SV-specific protein antibodies, SV2 and VAMP-2.

22 POROSOME: THE UNIVERSAL SECRETORY MACHINERY IN CELLS

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